Postremission MRD
monitoring for detection of an MRD relapse
Do you conduct
additional MRD assessments based on individual clinical decisions?

At what intervals per
patient do you perform post-remission MRD assessment for detection of an
MRD relapse?

| Clinical Signs |
prolonged cytopenias |
| Clinical Signs |
Standard risk (after TP1)/Medium risk patients (after
TP2): Only if there are clincal or laboratory signs of relapse
(e.g. prolonged cytopenia…) |
| Fixed Intervals |
Every 3-4 months |
| Fixed Intervals |
evry three months at 1st year, every six months for the
2nd year and then yearly up to 5 years |
| Fixed Intervals |
Ph negative ALL, chemotherapy: first year: prior to
each consolidation cycle, second+third year: every 3 months, fourth
year: evey 6 months. Ph negative ALL, after SCT: every 3 months. Ph+
ALL, after SCT: every 6 weeks in blood, every 3 months in BM. |
| Fixed Intervals |
every 3 months (three years)/ 2 months (2 years), and
if there are clinical signs of relapse |
| Fixed Intervals |
3 |
| Fixed Intervals |
depends on a situation, follow up post-SCT is usually
day 30, day 60, day 180, 1 yr, 2yr, 3yr, in relapsed based on relapse
protocol |
| Fixed Intervals |
end of therapy |
| Fixed Intervals |
MRD post alloHSCT (d+30, d+60, d+100, d+180,
d+365) |
| Other |
If there are clinical/lab signs suspicious for relapse,
and in pts. after alloHSCT |
| Other |
after each treatment block, ad hoc practice in
maintenance every 3 months |
| Other |
MRD after each element of HR-Therapy (AIEOP-BFM:all
HR-patients: TPHR1, TPHR2, TPHR3 (=MRD after each HR-Block),
additionally all HR-T-ALL, all Infants, B-ALL (not atl east 2 x negative
in subsequent time points): prior to Re-Induction (prior to 2. Protocol
III and 3. Protocol III) |
Statement 3: Postremission MRD assessment
should be performed at fixed intervals according to the protocol and
additionally in response to clinical signs of relapse.
What are the relevant
MRD cut-off values used for relapse detection in Ph-negative
ALL?

| Other |
Positivity below QR: warning signal, MRD >1XE-04:
molecular relapse |
| Other |
5x10-2 |
| Other |
any MRD positivity, but in positive not quantifiable
verified by NGS |
| Other |
Any MRD-positivity with further confirmation with other
method and different sample |
| Other |
We follow PDL guidelines |
| Other |
For relapse definition more than 1 method is needed for
confirmation, if MRD is < 5%. MRD of 5E-04 is highly suspicious of
pending relapse if detected late in therapy |
Statement 6: For postremission relapse
detection, any MRD positivity is considered relevant; however, values
below 1×10⁻⁴ should be confirmed using an additional method and a second
sample.
What are the relevant
IG/TR-based MRD cut-off values used for relapse
detection in Ph-positive ALL?

| Other |
not fully standardized, probably like in Ph-negative
ALL. |
| Other |
5x10-2 |
| Other |
we do BCR/ABL on DNA level simultaneously and solve
individually (CML-like cases) |
| Other |
Any MRD-positivity with further confirmation with other
method and different sample |
| Other |
We follow PDL guidelines |
| Other |
do not use IG/TR-MRD; 1x10-2 for flow |
| Other |
see question 18 |
What are the relevant
BCR::ABL1-based MRD cut-off values used for relapse
detection in Ph-positive ALL?

| Other |
Likely not relevant with IG/TR testing |
| Other |
none |
| Other |
reported in the context of simultaneous measurement of
DNA-based BCR/ABL and Ig/TR (e.g. BCR/ABL 1E-02 and Ig/TR negative does
not need to be relapse in CML-like cases) |
| Other |
ALLIC group did not define MRD cut-off value for
diagnosing molecular relapse in Ph+ALL. LOQ in CML is 1e-05 |
| Other |
Any MRD-positivity with further confirmation with other
method and different sample |
| Other |
We follow PDL guidelines |
| Other |
BCR::ABL1 positivity without Ig/TCR positivity and
zytomorphological absence of blasts is not considered a relapse (this
constellation is suspicious for misdiagnosis in the beginning –> CML
in blasts crisis ) |
Relapse
confirmation
Is a conversion to
MRD-positivity considered equivalent to relapse?

| Yes, always |
Yes if converts from negative to positive but only if
there is high confidence that the finding reflects true MRD (e.g. not
“background”), ideally confirmed on a follow-up sample. |
| Dependes on MRD level |
If ther is higher than 1x10e-4, confirmed in a second
sample |
| Dependes on MRD level |
1xE-04 |
| Dependes on MRD level |
if >0,01% |
| Dependes on MRD level |
10-4 but this is defined then as molecular relapse
which is different from hematologic relapse |
| Dependes on MRD level |
Clearly if 1E-02, but it would trigger clinical actions
if 1E-04 if confirmed by 2 tecniques in 2 different consecutive
samples |
| Dependes on MRD level |
1x10-2 |
| Dependes on MRD level |
one log increase required |
| Dependes on MRD level |
MRD 1 -<5%: two other tests with >=1% or second
BM. MRD <25% one additional test >= 1%. doi:
10.1182/blood.2021012328. PMID: 34192312; PMCID: PMC8952186. |
| No, never |
depends on ALL type-different situation in CML-like
ALL, see above. In Ph-neg ALL pos NQ after negative always verified by
NGS |
| No, never |
Only if MRD exceedes the 1% (1–02) limit for CR as
defined by the P-d-L paper |
| Depends on additional findings |
MRD-positivity conversion should be unambiguous: either
high level, or if low, without any doubt (confirmed on a second sample
or a second marker or technique, or with clinical signs of
extramedullary relapse)) |
| Depends on additional findings |
Flow or/and morphology |
Statement 7: Conversion to MRD positivity
should be considered a relapse only if the MRD level exceeds 1×10⁻⁴ and
is confirmed by an additional method or other molecular target.
Should the conversion
from IG/TR-based MRD-negativity to MRD-positivity be
confirmed by a consecutive sample?

| Other |
depends on the context and MRD level |
| Other |
We follow PdL so depends on if a confirmatory test and
level of detection |
| Other |
depends on the level <10-4 likely requires
repeat |
| Other |
No, a single positive result is sufficient: depends on
the Level (see 21) |
Statement 8: Conversion from IG/TR-based
MRD-negativity to MRD-positivity should be confirmed by a consecutive
sample only if the MRD level is below 1×10⁻⁴.
Should the conversion
from BCR::ABL1-based MRD-negativity to MRD-positivity
be confirmed by a consecutive sample?

| Other |
Only if accompanied by MRD by IG/TR |
| Other |
IG/TR to be analyzed |
| Other |
Relapse should be confirmed by IG/TR MRD in bone
marrow |
| Other |
in CML like completely different situation, usually
repeated sample necessary |
| Other |
We follow PdL |
| Other |
no routine BCR::ABL1 monitoring performed |
Statement 9: Conversion from
BCR::ABL1-based MRD-negativity to MRD-positivity should be considered a
relapse only if accompanied by IG/TR-based MRD positivity.
If MRD conversion
requires confirmation, within what time frame should the consecutive
sample be taken?

| Other |
No clear rule |
| Other |
2 weeks |
| Other |
We follow PdL |
| Other |
<1 week for positive >1% without morphology, 2-4
weeks for lower level |
Statement 10: If MRD conversion requires
confirmation, the consecutive sample should be taken within 1–4 weeks.
Bone marrow
sampling
In the clinical study
your patients are treated in, is there a standard operation procedure
(SOP) for bone marrow sampling?

| Other |
There is a manual that states: 4.6.3 Material 10 ml
Knochenmark und/oder 10 ml peripheres Blut Von jedem
Untersuchungszeitpunkt, möglichst 1. Aspirat EDTA bevorzugt, Heparin
auch möglich Die Zeitpunkte der Remissionskontrollen und Einsendung von
Probenmaterial zur MRD-Untersuchung sind den aktuellen Protokollen bzw.
Therapieempfehlungen zu entnehmen. Praktisch wird die KM-Punktion
jeweils nach Regeneration durchgeführt. Es handelt sich also nicht um
Aplasie-Punktionen. In addition, we hav a local SOP in Kiel |
| Other |
instructions are give in an extra document of the
sample request form |
Apart from its use in
diagnosing and classifying ALL, how else is diagnostic bone marrow
aspirate utilized in clinical or research settings?

| Other |
research on add-on projects |
| Other |
For pilot research purposes |
| Other |
molecular genetics |
Statement 11: Beyond diagnosis and
classification, diagnostic bone marrow aspirates should be used for LAIP
identification, molecular MRD marker screening, as reference material
for follow-up analyses, and biobanking.
Which pull of the
bone marrow aspiration is used for MRD detection during follow-up?

If the first pull of
bone marrow aspiration is not used for MRD assessment, what is it
typically used for?

Statement 12: The first pull of bone
marrow aspiration should be used for MRD assessment, as hemodilution in
later pulls may hamper accurate MRD quantification.
What collection
system do you use for bone marrow aspiration?

| Other |
See Kiel SOP |
| Other |
Don’t know if it is standardized |
| Other |
do not know and I am afraid to go ask somewhere because
it seems impossible to save the answers and I would loose too much
work |
| Other |
systems differ between clinical departments |
What is the volume of
the collection system you use for bone marrow aspiration?

| Other |
not applicable |
| Other |
2-5 ml left and 2-5 ml right |
| Other |
depending of age and collection system all of the
answers above |
What volume of bone
marrow do you typically aspirate during the procedure?

| Other |
in multiple aspirates |
| Other |
Following the SOP , we perform 2 aspirates of 4ml (1st
aspirate) and 10ml (2nd aspirate at different angle through same skin
puncture) |
| Other |
we do repeat pulls of approximately 2ccs with
reanchoring |
| Other |
depending of age and collection system all of the
answers above |
What volume of bone
marrow do you typically provide for MRD analysis?

| Other |
we receive DNA already extracted |
| Other |
Optimally 2.0 mL |
| Other |
depends on the cell number |
| 6–10 mL |
depending of age and collection system all of the
answers above |
Statement 13: Up to 10mL of bone marrow
should be aspirated in the first pull, of which 2–5mL should be provided
for MRD analysis.
Sample
preparation
Are your bone marrow
samples being checked for hemodilution?

| No, never |
We send the sample to the central lab for FCM and there
check for hemodilution |
| Yes, using Cytomorphology |
Presence/absence of bone marrow particles |
| Yes, using Cytomorphology |
assessment of spicules |
| Yes, using Cytomorphology |
Important for remission evaluation at day 33 (TP1) |
| Yes, using Flow cytometry |
checked for but no calculation, remark made in
conclusion |
| Yes, using Flow cytometry |
do not know and do not want to go ask FC guys because
it seems impossible to save the answers in the process. can send
later |
| Yes, using Flow cytometry |
Presence of <2% of EBL |
| Yes, using Flow cytometry |
Only day 15: Important for FCM-MRD evaluation |
Are your bone marrow
samples being processed using Ficoll separation prior to MRD
analysis?

Statement 20: Bone marrow samples should
be processed using Ficoll separation prior to molecular MRD analysis.
When preparing the
first dilution step (typically 1E-01) for the standard curve from a
Ficoll-processed sample, do you take the blast percentage into
account?

| Other |
yes, when blasts are below 50% |
| Other |
yes if it is below 50% |
| Other |
this varies based on each center practice |
| Other |
we adjust the standard curve according to
cytomorphology, if blast infiltration is below 50% however we are trying
to implement Flow after Ficoll at the moment in order to be more
exact |
Statement 21: When preparing the first
dilution step (typically 1×10⁻¹) for the standard curve from a
Ficoll-processed sample, the blast percentage should always be taken
into account.
Technical requirements
flow cytometry
Which anticoagulant
do you use for bone marrow samples intended for flow-cytometry MRD
analysis?

Statement 25: Bone marrow samples
intended for flow cytometry MRD analysis should be collected using
either heparin or EDTA as an anticoagulant.
Do you apply bulk
lysis to your samples?

| NA |
I do not know |
| Only at certain time points (please specify) |
Not in aplasia |
| Only at certain time points (please specify) |
in case of low cellularity |
| NA |
Sometimes |
| Only at certain time points (please specify) |
only remission samples (day 33 onwards), not day 15 and
not inital diagnosis |
Statement 26: Bulk lysis should be
applied to bone marrow samples prior to flow cytometry analysis.
What is the minimum
number of cells you typically measure?

| Other |
I do not know ( I am clinician) |
| Other |
Depends on the time-point. In ALL-BFM, we do day 15
MRD, and there we acquire 500,000 cells. In GMALL and in ALL-BFM at
other time-points, we aim for 4,000,000 events. |
| Other |
Dx 100,000, day 15 100,000-500,000, day 33 more than
1,000,000 |
| Other |
500,000 cells |
| Other |
Varies based on lab and method |
| Other |
remission spamples:1-10. Mio, Initial + day 15 :
100.000-500,000 |
What is the minimum
number of events required to define MRD positivity (LOD)?

| Other |
do not know ( I am clinician) |
| Other |
10 events |
| Other |
Varies based on lab and method |
Statement 27: A minimum of 10–20 events
is required to define MRD positivity (limit of detection) in flow
cytometry analysis.
What is the minimum
number of events required to quantify MRD (LOQ)?

| NA |
do not know ( I am clinician) |
| Other |
40 events |
| Other |
Varies based on lab and method |
Statement 28: A minimum of 10–20 events
is required to quantify MRD (limit of quantification) in flow cytometry
analysis.
Statement 29: A minimum of 21-50 events
is required to quantify MRD (limit of quantification) in flow cytometry
analysis.
What level of
sensitivity (LOD) is typically achieved in your MRD analysis?

| Other |
BCP-ALL: E-05, T-ALL: rather E-04 or less |
| Other |
depends on timepoint, see above |
| Other |
2X10-5 |
| Other |
It varies according to the time point |
| Other |
day 15: 1E-03 (without bulk lyisis) |
Statement 30: Flow cytometry MRD analysis
should achieve a sensitivity of at least 1×10⁻⁴
Statement 31: Flow cytometry MRD analysis
should achieve a sensitivity of at least 5×10⁻⁵
Statement 32: Flow cytometry MRD analysis
should achieve a sensitivity of at least 1×10⁻⁵
Statement 33: Flow cytometry MRD analysis
should achieve a sensitivity of at least 1×10⁻⁶
What level of
quantification (LOQ) is typically achieved in your MRD analysis?

| Other |
BCP-ALL: 5xE-05, T-ALL: rather E-04 or less |
| Other |
day 15 0.1%, day 33 0.1%-0.01% |
| Other |
6X10-5 |
| Other |
It varies according to the time point |
Statement 34: Flow cytometry MRD analysis
should achieve a limit of quantification of at least 1×10⁻⁴
Statement 35: Flow cytometry MRD analysis
should achieve a limit of quantification of at least 5×10⁻⁵
Statement 36: Flow cytometry MRD analysis
should achieve a limit of quantification of at least 1×10⁻⁵
Statement 37: Flow cytometry MRD analysis
should achieve a limit of quantification of at least 1×10⁻⁶
Do you use a
commercial assay or an in-house assay for your MRD analysis?

| Commercial |
NSG MRD is commercial |
| Commercial |
ClonoSeq for NGS-based |
| EuroFlow |
BCP-ALL: EuroFlow, but as in-house assay. T-ALL:
in-house assay according to BFM-Flow SOP |
| EuroFlow |
BCP-ALL MRD assay |
| EuroFlow |
BD CytognosTM B-cell Precursors ALL MRD |
| EuroFlow |
B-MRD plus 1 additional in-house tube |
| In-house |
I-BFM standard |
| In-house |
Flow is both performed locally and centrally and
depends on population and trial and timepoints |
| In-house |
St. Jude in house |
| In-house |
T-MRD |